Novel apoptosis-modulating proteins, dna encoding the proteins and methods of use thereof

ABSTRACT

The present invention provides a novel family of apoptosis-modulating proteins. Nucleotide and amino acid residue sequences and methods of use thereof are also provided.

This is a continuation-in-part of U.S. patent application Ser. No.08/160,067 filed Nov. 30, 1993.

FIELD OF THE INVENTION

This invention relates to novel proteins with apoptosis-modulatingactivity, recombinant DNA encoding the proteins, compositions containingthe proteins and methods of use thereof.

BACKGROUND OF THE INVENTION

Apoptosis is a normal physiologic process that leads to individual celldeath. This process of programmed cell death is involved in a variety ofnormal and pathogenic biological events and can be induced by a numberof unrelated stimuli. Changes in the biological regulation of apoptosisalso occur during aging and are responsible for many of the conditionsand diseases related to aging. Recent studies of apoptosis have impliedthat a common metabolic pathway leading to cell death may be initiatedby a wide variety of signals, including hormones, serum growth factordeprivation, chemotherapeutic agents, ionizing radiation and infectionby human immunodeficiency virus (HIV). Wyllie (1980) Nature,284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun.118:392-399; Duke and Cohen -(1986) Lymphokine Res. 5:289-299; Tomei etal. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al.(1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991)Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143.Agents that modulate the biological control of apoptosis thus havetherapeutic utility in a wide variety of conditions.

Apoptotic cell death is characterized by cellular shrinkage, chromatincondensation, cytoplasmic blebbing, increased membrane permeability andinterchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; andCohen and Duke (1992) Ann. Rev. Immunol. 10:267. The blebs, small,membrane-encapsulated spheres that pinch off of the surface of apoptoticcells, may continue to produce superoxide radicals which damagesurrounding cell tissue and may be involved in inflammatory processes.

Bcl-2 was discovered at the common chromosomal translocation sitet(14:18) in follicular lymphomas and results in aberrant over-expressionof bcl-2. Tsujimoto et al. (1984) Science 226:1097-1099; and Cleary etal. (1986) Cell 47:19-28. The normal function of bcl-2 is the preventionof apoptosis; unregulated expression of bcl-2 in B cells is thought tolead to increased numbers of proliferating B cells which may be acritical factor in the development of lymphoma. McDonnell and Korsmeyer(1991) Nature 349:254-256; and, for review see, Edgington (1993)Bio/Tech. 11:787-792. Bcl-2 is also capable of blocking of γirradiation-induced cell death. Sentman et al. (1991) Cell 67:879-888;and Strassen (1991) Cell 67:889-899. It is now known that bcl-2 inhibitsmost types of apoptotic cell death and is thought to function byregulating an antioxidant pathway at sites of free radical generation.Hockenbery et al. (1993) Cell 75:241-251.

While apoptosis is a normal cellular event, it can also be induced bypathological conditions and a variety of injuries. Apoptosis is involvedin a wide variety of conditions including but not limited to,cardiovascular disease, cancer regression, immunoregulation, viraldiseases, anemia, neurological disorders, gastrointestinal disorders,including but not limited to, diarrhea and dysentery, diabetes, hairloss, rejection of organ transplants, prostate hypertrophy, obesity,ocular disorders, stress and aging.

Bcl-2 belongs to a family of proteins some of which have been cloned andsequenced. Williams and Smith (1993) Cell 74:777-779. All referencescited herein, both supra and infra, are hereby incorporated by referenceherein.

SUMMARY OF THE INVENTION

Substantially purified DNA encoding novel bcl-2 homologs, termed cdn-1,cdn-2 and cdn-3, as well as recombinant cells and transgenic animalsexpressing the cdn-1 and cdn-2 genes are provided. The substantiallypurified CDN-1 and CDN-2 proteins and compositions thereof are alsoprovided. Diagnostic and therapeutic methods utilizing the DNA andproteins are also provided. Methods of screening for pharmaceuticalagents that stimulate, as well as pharmaceutical agents that inhibitcdn-1 and cdn-2 activity levels are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the PCR primers used to isolate the cdn-1 probes.

FIG. 2 depicts the cdn-1 clones obtained by the methods described inExample 1.

FIG. 3 depicts the nucleotide sequence of cdn-1.

FIG. 4 depicts the results of a Northern blot analysis of multipletissues with probes specific for both bcl-2 and cdn-1.

FIG. 5 shows the sequence of the cdn-2 cDNA and flanking sequences andthe corresponding predicted amino acid sequence of the cdn-2 protein.

FIG. 6 shows a comparison of N-terminal amino acid sequences of cdn-1,cdn-2 and known bcl-2 family members.

FIG. 7 shows the nucleotide sequence of cdn-3.

FIG. 8 shows the anti-apoptotic effects of cdn-1 and some of itsderivatives in serum-deprivation induced apoptosis of WIL-2 cells.

FIG. 9 shows anti-apoptotic effects of cdn-1 and some of its derivativesin FAS-induced apoptosis of WIL-2 cells.

FIG. 10 shows modulation of apoptosis by cdn-1 and cdn-2 in FL5.12cells.

FIG. 11 depicts the cdn-1 derivative proteins Δ1, Δ2 and Δ3. TheN-terminal residues are indicated by the arrows. The remainder of thederivative proteins is the same as full-length cdn-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses substantially purified nucleotidesequences encoding the novel bcl-2 homologs, cdn-1 and cdn-2; and theproteins encoded thereby; compositions comprising cdn-1 and cdn-2 genesand proteins and methods of use of thereof. Note that in copending U.S.patent application Ser. No. 08/160,067, cdn-1 was termed cdi-1; althoughthe name has been changed, the nucleotide sequence remains identical.The invention further includes recombinant cells and transgenic animalsexpressing the cloned cdn-1 or cdn-2 genes. The nucleotide and predictedamino acid residue sequences of cdn-1 are shown in FIG. 3; and those ofcdn-2 are shown in FIG. 5. It has now been found that the proteinsencoded by the cdn genes are capable of modulating apoptosis. In alymphoblastoid cell line, cdn-1 was shown to decrease Fas-mediatedapoptosis. In a mouse progenitor B cell line, FL5.12, cdn-2 and aderivative of cdn-1 decrease IL-3-induced apoptosis whereas cdn-1slightly increased apoptosis. Thus, depending on the cell type, thederivative of cdn and the method of induction of apoptosis, apoptosiscan be modulated in a highly specific manner by controlling theconcentration of cdns.

As used herein, “cdns” or “cdn” refers to the nucleic acid moleculesdescribed herein (cdn-1, cdn-2, cdn-3 and derivatives thereof), “theCDNs” or “CDN” refers to the proteins encoded thereby (CDN-1, CDN-2,CDN-3 and derivatives thereof). The present invention encompasses cdn-1and cdn-2 nucleotide sequences. The cdn nucleotides include, but are notlimited to, the cDNA, genome-derived DNA and synthetic or semi-syntheticDNA or RNA. The nucleotide sequence of the cdn-1 cDNA with the locationof restriction endonuclease sites is shown in FIG. 2. As described inthe examples herein, cdn-1 mRNA has been detected in a variety of humanorgans and tissues by Northern blot analysis. These organs includeliver; heart; skeletal muscle; lung; kidney; and pancreas as shown inFIG. 3.

Similarly, cdn-2, cdn cDNA, genomic DNA and synthetic or semi-syntheticDNAs and RNAs are additional embodiments of the present invention. Thenucleotide sequence of cdn-2 cDNA, along with the predicted amino acidsequence of cdn-2 protein and the locations of restriction endonucleaserecognition sites, is given in FIG. 5. The examples presented hereinindicate that cdn-1 is on human chromosome 6 and that cdn-2 is on humanchromosome 20. There is also a member of the family cdn-3 which is onhuman chromosome 11. Fluorescence in situ hybridization (FISH) indicatedan approximate location of cdn-1 to be at 6p21-23. Within this regionresides the gene for spinocerebellar ataxia type 1. Interestingly,apoptosis has been proposed recently to be involved in the relatedgenetic disorder ataxia telangiectasia. Taken together with thechromosomal localization and the expression of cdn-1 in brain tissue,this suggests the possibility that cdn-1/cdn-2 might represent the SCA1gene locus. It is possible that cdn-2 and cdn-3 are pseudogenes. Whilethese may not be expressed endogenously, they are capable of expressionfrom a recombinant vector providing the appropriate promoter sequences.Thus, both cdn-2 and cdn-3 genes are encompassed by the presentinvention as are recombinant constructs thereof and proteins encodedthereby.

Derivatives of the genes and proteins include any portion of theprotein, or gene encoding the protein, which retains apoptosismodulating activity. FIG. 10 depicts three such derivatives of cdn-1which have been shown to retain apoptosis-modulating activity. Thesederivatives, cdn1-Δ1, cdn1-Δ2 and cdn1-Δ3, are encompassed by thepresent invention.

The invention includes modifications to cdn DNA sequences such asdeletions, substitutions and additions particularly in the non-codingregions of genomic DNA. Such changes are useful to facilitate cloningand modify gene expression.

Various substitutions can be made within the coding region that eitherdo not alter the amino acid residues encoded or result in conservativelysubstituted amino acid residues. Nucleotide substitutions that do notalter the amino acid residues encoded are useful for optimizing geneexpression in different systems. Suitable substitutions are known tothose of skill in the art and are made, for instance, to reflectpreferred codon usage in the particular expression systems.

The invention encompasses functionally equivalent variants andderivatives of cdns which may enhance, decrease or not significantlyaffect the properties of CDNs. For instance, changes in the DNA sequencethat do not change the encoded amino acid sequence, as well as thosethat result in conservative substitutions of amino acid residues, one ora few amino acid deletions or additions, and substitution of amino acidresidues by amino acid analogs are those which will not significantlyaffect its properties.

Amino acid residues which can be conservatively substituted for oneanother include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine. Anyconservative amino acid substitution which does not significantly affectthe properties of CDNs is encompassed by the present invention.

Techniques for nucleic acid manipulation useful for the practice of thepresent invention are described in a variety of references, includingbut not limited to, Molecular Cloning: A Laboratory Manual, 2nd ed.,Vol. 1-3, eds. Sambrook et al. Cold Spring Harbor Laboratory Press(1989); and Current Protocols in Molecular Biology, eds. Ausubel et al.,Greene Publishing and Wiley-Interscience: New York (1987) and periodicupdates.

The invention further embodies a variety of DNA vectors having clonedtherein the cdn nucleotide sequences encoding. Suitable vectors includeany known in the art including, but not limited to, those for use inbacterial, mammalian, yeast and insect expression systems. Specificvectors are known in the art and need not be described in detail herein.

The vectors may also provide inducible promoters for expression of thecdns. Inducible promoters are those which do not allow constitutiveexpression of the gene but rather, permit expression only under certaincircumstances. Such promoters may be induced by a variety of stimuliincluding, but not limited to, exposure of a cell containing the vectorto a ligand, metal ion, other chemical or change in temperature.

These promoters may also be cell-specific, that is, inducible only in aparticular cell type and often only during a specific period of time.The promoter may further be cell cycle specific, that is, induced orinducible only during a particular stage in the cell cycle. The promotermay be both cell type specific and cell cycle specific. Any induciblepromoter known in the art is suitable for use in the present invention.

The invention further includes a variety of expression systemstransfected with the vectors. Suitable expression systems include butare not limited to bacterial, mammalian, yeast and insect. Specificexpression systems and the use thereof are known in the art and are notdescribed in detail herein.

The invention encompasses ex vivo transfection with cdns, in which cellsremoved from animals including man are transfected with vectors encodingCDNs and reintroduced into animals. Suitable transfected cells includeindividual cells or cells contained within whole tissues. In addition,ex vivo transfection can include the transfection of cells derived froman animal other than the animal or human subject into which the cellsare ultimately introduced. Such grafts include, but are not limited to,allografts, xenografts, and fetal tissue transplantation.

Essentially any cell or tissue type can be treated in this manner.Suitable cells include, but are not limited to, cardiomyocytes andlymphocytes. For instance, lymphocytes, removed, transfected with therecombinant DNA and reintroduced into an HIV-positive patient mayincrease the half-life of the reintroduced T cells.

As an example, in treatment of HIV-infected patients by theabove-described method, the white blood cells are removed from thepatient and sorted to yield the CD4⁺ cells. The CD4⁺ cells are thentransfected with a vector encoding CDNs and reintroduced into thepatient. Alternatively, the unsorted lymphocytes can be transfected witha recombinant vector having at least one cdn under the control of acell-specific promoter such that only CD4⁺ cells express the cdn genes.In this case, an ideal promoter would be the CD4 promoter; however, anysuitable CD4⁺ T cell-specific promoter can be used.

Further, the invention encompasses cells transfected in vivo by thevectors. Suitable methods of in vivo transfection are known in the artand include, but are not limited to, that described by Zhu et al. (1993)Science 261:209-211. In vivo transfection by cdns may be particularlyuseful as a prophylactic treatment for patients suffering fromatherosclerosis. Elevated modulation of the levels of CDN could serve asa prophylaxis for the apoptosis-associated reperfusion damage thatresults from cerebral and myocardial infarctions. In these patients witha high risk of stroke and heart attack, the apoptosis and reperfusiondamage associated with arterial obstruction could be prevented or atleast mitigated.

Infarctions are caused by a sudden insufficiency of arterial or venousblood supply due to emboli, thrombi, or pressure that produces amacroscopic area of necrosis; the heart, brain, spleen, kidney,intestine, lung and testes are likely to be affected. Apoptosis occursto tissues surrounding the infarct upon reperfusion of blood to thearea; thus, modulation of CDN levels, achieved by a biologicalmodifier-induced change in endogenous production or by in vivotransfection, could be effective at reducing the severity of damagecaused by heart attacks and stroke.

Transgenic animals containing the recombinant DNA vectors are alsoencompassed by the invention. Methods of making transgenic animals areknown in the art and need not be described in detail herein. For areview of methods used to make transgenic animals, see, e.g. PCTpublication No. WO 93/04169. Preferably, such animals expressrecombinant cdns under control of a cell-specific and, even morepreferably, a cell cycle specific promoter.

In another embodiment, diagnostic methods are provided to detect theexpression of cdns either at the protein level or the mRNA level. Anyantibody that specifically recognizes CDNs is suitable for use in CDNdiagnostics. Abnormal levels of CDNs are likely to be found in thetissues of patients with diseases associated with inappropriateapoptosis; diagnostic methods are therefore useful for detecting andmonitoring biological conditions associated with such apoptosis defects.Detection methods are also useful for monitoring the success ofCDN-related therapies.

Purification or isolation of CDNs expressed either by the recombinantDNA or from biological sources such as tissues can be accomplished byany method known in the art. Protein purification methods are known inthe art. Generally, substantially purified proteins are those which arefree of other, contaminating cellular substances, particularly proteins.Preferably, the purified CDNs are more than eighty percent pure and mostpreferably more than ninety-five percent pure. For clinical use asdescribed below, the CDNs are preferably highly purified, at least aboutninety-nine percent pure, and free of pyrogens and other contaminants.

Suitable methods of protein purification are known in the art andinclude, but are not limited to, affinity chromatography, immunoaffinitychromatography, size exclusion chromatography, HPLC and FPLC. Anypurification scheme that does not result in substantial degradation ofthe protein is suitable for use in the present invention.

The invention also includes the substantially purified CDNs having theamino acid residue sequences depicted in FIGS. 3 and 5, respectively.The invention encompasses functionally equivalent variants of CDNs whichdo not significantly affect their properties and variants which retainthe same overall amino acid sequence but which have enhanced ordecreased activity. For instance, conservative substitutions of aminoacid residues, one or a few amino acid deletions or additions, andsubstitution of amino acid residues by amino acid analogs are within thescope of the invention.

Amino acid residues which can be conservatively substituted for oneanother include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine. Anyconservative amino acid substitution which does not significantly affectthe properties of CDNs is encompassed by the present invention.

Suitable antibodies are generated by using the CDNs as an antigen or,preferably, peptides encompassing the CDN regions that lack substantialhomology to the other gene products of the bcl family. Methods ofdetecting proteins using antibodies and of generating antibodies usingproteins or synthetic peptides are known in the art and are not bedescribed in detail herein.

CDN protein expression can also be monitored by measuring the level ofcdn MRNA. Any method for detecting specific mRNA species is suitable foruse in this method. This is easily accomplished using the polymerasechain reaction (PCR). Preferably, the primers chosen for PCR correspondto the regions of the cdn genes which lack substantial homology to othermembers of the bcl gene family. Alternatively, Northern blots can beutilized to detect cdn mRNA by using probes specific to cdns. Methods ofutilizing PCR and Northern blots are known in the art and are notdescribed in detail herein.

Methods of treatment with cdns also include modulating cellularexpression of cdns by increasing or decreasing levels of cdn mRNA orprotein. Suitable methods of increasing cellular expression of cdninclude, but are not limited to, increasing endogenous expression andtransfecting the cells with vectors encoding cdns. Cellular transfectionis discussed above and is known in the art. Suitable indications forincreasing endogenous levels of cdn include, but are not limited to,malignancies and cardiac-specific over-expression. Cardiac specificover-expression is particularly suitable for use in indicationsincluding, but not limited to, patients susceptible to heart disease andin advance of cardiotoxic therapies including, but not limited to,chemotherapies such as adriamycin, so as to offer cardioprotection.

In addition, increasing endogenous expression of cdns can beaccomplished by exposing the cells to biological modifiers that directlyor indirectly increase levels of CDNs either by increasing expression orby decreasing degradation of cdn mRNA. Suitable biological modifiersinclude, but are not limited to, molecules and other cells. Suitablemolecules include, but are not limited to, drugs, cytokines, smallmolecules, hormones, combinations of interleukins, lectins and otherstimulating agents e.g. PMA, LPS, bispecific antibodies and other agentswhich modify cellular functions or protein expression. Cells are exposedto such biological modifiers at physiologically effectiveconcentrations, and the expression of cdns is measured relative to acontrol not exposed to the biological modifiers. Those biologicalmodifiers which increase expression of cdns relative to the control areselected for further study.

The invention further encompasses a method of decreasing endogenouslevels of cdns. The methods of decreasing endogenous levels of cdnsinclude, but are not limited to, antisense nucleotide therapy anddown-regulation of expression by biological modifiers. Antisense therapyis known in the art and its application will be apparent to one of skillin the art.

Screening for therapeutically effective biological modifiers is done byexposing the cells to biological modifiers which may directly orindirectly decrease levels of CDNs either by decreasing expression or byincreasing the half-life of cdn mRNA or CDNs. Suitable biologicalmodifiers include, but are not limited to, molecules and other cells.Suitable molecules include, but are not limited to, drugs, cytokines,small molecules, hormones, combinations of interleukins, lectins andother stimulating agents e.g. PMA, LPS, bispecific antibodies and otheragents which modify cellular functions or protein expression. Cells aregrown under conditions known to elicit expression of at least one cdn(preferably cdn-1), exposed to such biological modifiers atphysiologically effective concentrations, and the expression of cdns ismeasured relative to a control not exposed to biological modifiers.Those biological modifiers which decrease the expression of cdnsrelative to a control are selected for further study. Cell viability isalso monitored to ensure that decreased cdn expression is not due tocell death.

In determining the ability of biological modifiers to modulate (increaseor decrease) cdn expression, the levels of endogenous expression may bemeasured or the levels of recombinant fusion proteins under control ofcdn-specific promoter sequences may be measured. The fusion proteins areencoded by reporter genes.

Reporter genes are known in the art and include, but are not limited tochloramphenicol acetyl transferase (CAT) and β-galactosidase. Expressionof cdn-1 and -2 can be monitored as described above either by protein ormRNA levels. Expression of the reporter genes can be monitored byenzymatic assays, or antibody-based assays, like ELISAs and RIAs, alsoknown in the art. Potential pharmaceutical agents can be any therapeuticagent or chemical known to the art, or any uncharacterized compoundsderived from natural sources such as fungal broths and plant extracts.Preferably, suitable pharmaceutical agents are those lacking substantialcytotoxicity and carcinogenicity.

Suitable indications for modulating endogenous levels of cdns are any inwhich cdn-mediated apoptosis is involved. These include, but are notlimited to, various types of malignancies and other disorders resultingin uncontrolled cell growth such as eczema, or deficiencies in normalprogrammed cell death such as malignancies, including, but not limitedto, B cell lymphomas.

The invention also encompasses therapeutic methods and compositionsinvolving treatment of patients with biological modifiers to increase ordecreast expression of cdns. Effective concentrations and dosageregimens may be empirically derived. Such derivations are within theskill of those in the art and depend on, for instance, age, weight andgender of the patient and severity of the disease. Alternatively,patients may be directly treated with either native or recombinant CDNs.The CDNs should be substantially pure and free of pyrogens. It ispreferred that the recombinant CDNs be produced in a mammalian cell lineso as to ensure proper glycosylation. CDNs may also be produced in aninsect cell line and will be glycosylated.

For therapeutic compositions, a therapeutically effective amount ofsubstantially pure CDN is suspended in a physiologically accepted bufferincluding, but not limited to, saline and phosphate buffered saline(PBS) and administered to the patient. Preferably administration isintravenous. Other methods of administration include but are not limitedto, subcutaneous, intraperitoneal, gastrointestinal and directly to aspecific organ, such as intracardiac, for instance, to treat cell deathrelated to myocardial infarction.

Suitable buffers and methods of administration are known in the art. Theeffective concentration of a CDN will need to be determined empiricallyand will depend on the type and severity of the disease, diseaseprogression and health of the patient. Such determinations are withinthe skill of one in the art.

Bcl-2 is thought to function in an antioxidant pathway. Veis et al.(1993) Cell 75:229-240. Therefore, therapy involving CDNs is suitablefor use in conditions in which superoxide is involved. Administration ofCDNs results in an increased extracellular concentration of CDNs, whichis thought to provide a method of directly inhibiting superoxideaccumulation that may be produced by the blebs associated withapoptosis. The therapeutic method thus includes, but is not limited to,inhibiting superoxide mediated cell injury.

Suitable indications for therapeutic use of CDNs are those involvingfree radical mediated cell death and include, but are not limited to,conditions previously thought to be treatable by superoxide dismutase.Such indications include but are not limited to HIV infection,autoimmune diseases, cardiomyopathies, neuronal disorders, hepatitis andother liver diseases, osteoporosis, and shock syndromes, including, butnot limited to, septicemia.

Hybridization of cloned cdn DNA to messenger mRNA from various regionsof the brain indicated high levels of expression of cdn-1 in each of theregions studied (FIG. 8). Therefore, neurological disorders are anotherarea in which therapeutic applications of CDNs may be indicated.

The following examples are provided to illustrate but not limit thepresent invention. Unless otherwise specified, all cloning techniqueswere essentially as described by Sambrook et al. (1989) and all reagentswere used according to the manufacturer's instructions.

EXAMPLE 1 Identification and Cloning of cdn-1 cDNA

An amino acid sequence comparison of the six known bcl-2 family members(FIG. 6) revealed two regions with considerable sequence identity,namely amino acids 144-150 and 191-199. In an attempt to identify newbcl-2 family members, degenerate PCR primers based on sequences in theseregions were designed (FIG. 1) and PCR was performed using human heartcDNA and human B lymphoblastoid cell line (WIL-2) cDNA. PCR wasperformed using the Hot Start/Ampliwax technique (Perkin Elmer Cetus).The final concentration of the PCR primers and the template cDNA were 4μM and 0.1-0.2 ng/ml, respectively. The conditions for cDNA synthesiswere identical to those for first strand cDNA synthesis of the cDNAlibrary as described below. PCR was performed in a Perkin Elmer CetusDNA Thermal Cycler according to the method described by Kiefer et al.(1991) Biochem. Biophys. Res. Commun. 176:219-225, except that theannealing and extension temperatures during the first 10 cycles were 36°C. Following PCR, samples were treated with 5 units of DNA polymerase I,Klenow fragment for 30 min at 37° C. and then fractionated byelectrophoresis on a 7% polyacrylamide, 1 X TBE (Tris/borate/EDTA) gel.DNA migrating between 170-210 base pars was excised from the gel,passively eluted for 16 hours with gentle shaking in 10 mM Tris-HCl pH7.5, 1 mM EDTA (TE), purified by passage over an Elutip-D column(Schleicher and Schuell), ligated to the pCR-Script vector (Stratagene)and transformed into Escherichia coli strain XL1-Blue MRF (Stratagene).Plasmid DNA from transformants (white colonies) containing both theheart and WIL-2 PCR products was isolated using the Magic Miniprep DNAPurification System (Promega), and the DNA inserts were sequenced by thedideoxy chain termination method according to Sanger et al. (1977) Proc.Natl. Acad. Sci. USA 74:5463-5467 (USB, Sequenase version 2.0). DNAsequence analysis of the eleven heart PCR products revealed twosequences identical to bcl-x (Boise et al. (1993) Cell 74:597-608) andten other sequences unrelated to the bcl-2 family.

DNA sequence analyses of the eleven WIL-2 PCR products yielded one bcl-xsequence, five sequences identical to another bcl-2 family member, bax(Oldvai et al. (1993) Cell 74:609-619), four unrelated sequences and onenovel bcl-2 related sequence, termed cdn-1. The unique cdn-1 amino acidsequence encoded by the PCR product is shown in FIG. 6 from amino acid151-190 (top row).

To isolate the cdn-1 cDNA, a human heart cDNA library (Clontech) and aWIL-2 cDNA library, constructed as described by Zapf et al. (1990) J.Biol. Chem. 265:14892-14898 were screened using the cdn-1 PCR DNA insertas a probe. The DNA was ³²P-labeled according to the method described byFeinberg and Vogelstein (1984) Anal. Biochem. 137:266-267 and used toscreen 150,000 recombinant clones from both libraries according to themethod described by Kiefer et al. (1991). Eight positive clones from theWIL-2 cDNA library and two positive clones from the heart cDNA librarywere identified. Four clones from the WIL-2 cDNA library and two fromthe heart cDNA library were further purified and plasmid DNA containingthe cDNA inserts was excised from the λZAPII vector (Stratagene) (FIG.2). The two longest clones, W7 (2.1 kb) and W5 (2.0 kb) were sequencedand shown to contain the cdn-1 probe sequence, thus confirming theirauthenticity. The heart cDNAs also encoded cdn-1.

The W7 DNA sequence along with the deduced amino acid residue sequenceis shown in FIG. 2. The deduced amino acid sequence of cdn-1 was alsoaligned for maximum sequence identity with the other bcl-2 familymembers and is shown in FIG. 6. As can be seen, there is considerablesequence identity between cdn-1 and other family members between aminoacids 100 and 200. Beyond this central region, sequence conservationfalls off sharply. Like bcl-2, cdn-1 appears to be an intracellularprotein in that it does not contain a either a hydrophobic signalpeptide or N-linked glycosylation sites. Cdn-1 does contain ahydrophobic C-terminus that is also observed with all bcl-2 familymembers except LMW5-HL, suggesting its site of anti-apoptotic activity,like that of bcl-2, is localized to a membrane bound organelle such asthe mitochondrial membrane, the endoplasmic reticulum or the nuclearmembrane. Hockenbery et al. (1990); Chen-Levy et al. (1989) Mol. Cell.Biol. 9:701-710; Jacobsen et al. (1993) Nature 361:365-369; and Monighanet al. (1992) J. Histochem. Cytochem. 40:1819-1825.

EXAMPLE 2 Northern Blot Analysis of cDNA Clones

Northern blot analysis was performed according to the method describedby Lehrach et al. (1977) Biochem. 16:4743-4651 and Thomas (1980) Proc.Natl. Acad. Sci. USA 77:5201-5205. In addition, a human multiple tissueNorthern blot was purchased from Clontech. The coding regions of bcl-2and cdn-1 cDNAs were labeled by the random priming method described byFeinberg and Vogelstein (1984) Anal. Biochem. 137:266-267. Hybridizationand washing conditions were performed according to the methods describedby Kiefer et al. (1991).

The results, presented in FIG. 4 indicate that cdn-1 is expressed in allorgans tested (heart, brain, placenta, lung, liver, skeletal muscle,kidney and pancreas) whereas bcl-2 is not expressed or expressed at onlylow levels in heart, brain, lung, and liver. Thus, cdn-1 appears to bemore widely expressed throughout human organs than bcl-2 and may be moreimportant in regulating apoptosis in these tissues.

EXAMPLE 3 Expression of Recombinant cdn-1

In order to express recombinant cdn-1 in the baculovirus system, thecdn-1 cDNA generated in Example 1 was used to generate a novel cdn-1vector, by a PCR methodology as described in Example 1, using primersfrom the 3′ and 5′ flanking regions of the gene which containrestriction sites to facilitate cloning. The plasmids were sequenced bythe dideoxy terminator method (Sanger et al., 1977) using sequencingkits (USB, Sequenase version 2.0) and internal primers. This was toconfirm that no mutations resulted from PCR.

A clone was used to generate recombinant viruses by in vivo homologousrecombination between the overlapping sequences of the plasmid and AcNPVwild type baculovirus. After 48 hours post-transfection in insectSpodoptera frugiperda clone 9 (SF9) cells, the recombinant viruses werecollected, identified by PCR and further purified. Standard proceduresfor selection, screening and propagation of recombinant baculovirus wereperformed (Invitrogen). The molecular mass, on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), of the proteinproduced in the baculovirus system was compared with the predictedmolecular mass of cdn-1 according to the amino-acid sequence.

In addition, similar clones can be expressed preferably in a yeastintracellular expression system by any method known in the art,including the method described by Barr et al. (1992) Transgenesis, ed.JAH Murray, (Wiley and Sons) pp. 55-79.

EXAMPLE 4 Expression of cdn-1 in Mammalian Systems

The cdn-1 coding sequence was excised from a plasmid generated inExample 1, and introduced into plasmids pCEP7, pREP7 and pcDNA3(Invitrogen) at compatible restriction enzyme sites. pCEP7 was generatedby removing the RSV 3′-LTR of pREP7 with XbaI/Asp718, and substitutingthe CMV promoter from pCEP4 (Invitrogen). 25 μg of each cdn-1-containingplasmid was electroporated into the B lymphoblastoid cell line WIL-2,and stable hygromycin resistant transformants or G418 resistanttransformants (pcDNA3 constructs, FIG. 8) expressing cdn-1 wereselected.

The coding region of cdns can also ligated into expression vectorscapable of stably integrating into other cell types including but notlimited to cardiomyocytes, neural cell lines such as GTI-7 and TNFsensitive cells such as the human colon adenocarcinoma cell line HT29 soas to provide a variety of assay systems to monitor the regulation ofapoptosis by cdn-1.

EXAMPLE 5 Effect of the Anti-Apoptotic Activity of cdn-1 and itsDerivatives in the Wild Type B Lymphoblastoid Cell Line WIL2-729 HF2 andthe Transformed Cell Expressing Excess cdn-1

2×10⁵ WIL-2, and WIL-2 cells transformed with a vector encoding cdn-1 asdescribed in Example 4 are grown in RPMI supplemented with 10% fetalbovine serum (FBS) for the anti-fas experiment or 0.1% FBS for serumdeprivation experiments. In the case of the anti-fas experiment, afterwashing with fresh medium, the cells were suspended in RPMI supplementedwith 10% FBS, exposed to anti-fas antibodies and the kinetics of celldeath in response to an apoptosis inducing agent were analyzed by flowcytometry with FACScan. In the case of the serum deprivation experiment,the WIL-2 cells were resuspended in RPMI supplemented with 0.1% FBS andapoptosis was monitored according to the method described by Hendersonet al. (1993) Proc. Natl. Acad. Sci. USA 90:8479-8483. Other methods ofinducing apoptosis include, but are not limited to, oxygen deprivationin primary cardiac myocytes, NGF withdrawal, glutathione depletion inthe neural cell line GTI-7 or TNF addition to the HT29 cell line.Apoptosis was assessed by measuring cell shrinkage and permeability topropidium iodide (PI) during their death. In addition, any other methodof assessing apoptotic cell death may be used.

FIG. 8 shows the anti-apoptotic response of various WIL-2 transformantsto anti-Fas treatment. FIG. 9 shows the anti-apoptotic response ofvarious WIL-2 transformants to serum deprivation. In FIG. 8, duplicatewells containing 3×10⁵ cells were incubated with 50 ng/ml of thecytocidal anti-Fas antibody for 24 hours. Cell death was then analyzedby flow cytometry with FACScan. The proteins expressed from eachconstruct are shown beneath the columns. Since many of the constructsare truncation or deletion variants, the exact amino acids expressed arealso indicated. As can be seen, all of the transformants had someprotective effect when compared to the control transformant containingthe pREP7 vector alone. The most apoptosis-resistant transformant wasthe cdn-1Δ2 expressing cell line, in which over 90% of the cellssurvived anti-fas treatment. Significant protection was also observed intransformants expressing full length cdn-1 (1-211) and cdn-1Δ1, followedby bcl-2Δ and bcl-2 expressing cell lines.

Cdn-1Δ1 and cdn-1Δ2 are lacking the N-terminal 59 and 70 amino acids ofthe full length cdn-1 molecule, respectively. The observation thatcdn-1Δ2 is more effective at blocking apoptosis than full length cdn-1suggests that smaller, truncated cdn-1 molecules may be potenttherapeutics.

EXAMPLE 6 Determination of Other cdn Genes and Cloning of the cdn-2 Gene

Southern blot analyses of human genome DNA and a panel of human/rodentsomatic cell DNAs indicated that there were at least 3 cdn related genesand that they resided in chromosomes 6, 11 and 20. PCR/sequence analysisof the three hybrid DNAs showed that cdn-1 was on chromosome 6 and thattwo closely related sequences were on chromosome 20 (designated cdn-2)and chromosome 11 (designated cdn-3). We have cloned the cdn-2 and cdn-3genes and sequenced them. Interestingly, both cdn-2 and cdn-3 do notcontain introns and have all of the features of processed genes thathave returned to the genome. cdn-3 has a nucleotide deletion, causing aframe shift and early termination and thus is probably a pseudogene.Both, however, have promoter elements upstream of the repeats CCAAT,TATAAA boxes but are probably not transcribed. (Northern blot analysiswith cdn-2 and cdn-3 specified probes.)

900,000 clones from a human placenta genomic library in the cosmidvector pWE15 (Stratagene, La Jolla, Calif.) were screened with a 950 bpBgIII- HindIII cDNA probe containing the entire coding region of Cdn-1.The probe was ³²P-labeled according to the method of Feinberg andVogelstein (1984) Anal. Biochem. 137:266-267. The library was processedand screened under high stringency hybridization and washing conditionsas described by Sambrook et al. (1989) Molecular Cloning, 2nd edition,Cold Spring Harbor Laboratory Press. Ten double positive clones werefurther purified by replating and screening as above. Plasmid DNA waspurified using the Wizard Maxiprep DNA Purification System as describedby the supplier (Promega Corp., Madison, Wis.) and analyzed by EcoRIrestriction enzyme mapping and Southern blotting. The probe used forSouthern blotting and hybridization conditions was the same as above.

The cosmid clones fell into two groups as judged by EcoRI restrictionanalysis and Southern blotting. Cosmid clones (cos) 1-4 and 7 displayedone distinct pattern of EcoRI generated DNA fragments and contained asingle 6.5 kb hybridizing EcoRI DNA fragment. Cos2 and Cos9 fell intothe second group that was characterized by a 5.5 kb hybridizing EcoRIDNA fragment. The 6.5 kb DNA fragment from cos2 and the 5.5 kb DNAfragment from cos9 were subcloned into pBluescript SK (Stratagene, LaJolla, Calif.) using standard molecular biological techniques (Sambrooket al. as above). Plasmid DNA was isolated and the DNA inserts from twosubclones, A4 (from cos2) and C5 (from cos9) were mapped with BamHI,HindIII and EcoRI and analyzed by Southern blotting as described above.Smaller restriction fragments from both clones were subcloned into M13sequencing vectors and the DNA sequence was determined.

The sequence of A4 contains an open reading frame that displays 97%amino acid sequence identity with cdn-1. (FIG. 5) The high degree ofsequence identity of this gene with cdn-1 indicates that it is a newcdn-1 related gene and therefore will be called cdn-2. A sequencecomparison of the encoded cdn-2 protein and the other members of thebcl-2 family is shown in FIG. 5. Cdn-2 contains the conserved regions,BH1 and BH2, that are hallmarks of the bcl-2 family, and displays alower overall sequence identity (˜20-30%) to other members, which isalso characteristic of the bcl-2 family. cdn-3 has a frame shift andtherefore does not contain the structural features of cdn-1, cdn-2 orother bcl-2 family members.

EXAMPLE 7 Chromosomal Localization of the cdn-1 and cdn-2 Genes

Southern blot analysis of a panel of human/rodent somatic cell hybridDNAs (Panel #2 DNA from the NIGMS, Camden, N.J.) and fluorescent in situhybridization (FISH) of metaphase chromosomes were used to map the cdngenes to human chromosomes. For Southern blotting, 5μg of hybrid panelDNA was digested with EcoRI or BamHI/HindIII, fractionated on 0.8% or 1%agarose gels, transferred to nitrocellulose and hybridized with thecdn-1 probe. Hybridization and washing conditions were as describedabove. For FISH, the cdn-2 subclone, A4, was biotinylated using theBionick Labeling System (Gibco BRL, Gaithersburg, Md.) and hybridized tometaphase chromosomes from normal human fibroblasts according to themethod described by Viegas-Pequignot in In Situ Hybridization, APractical Approach, 1992, ed. D. G. Wilkinson, pp. 137-158, IRL Press,Oxford. Probe detection using FITC-conjugated avidin and biotinylatedgoat anti-avidin was according to the method described by Pinkel et al.(1988) Proc. Natl. Acad. Sci. USA 85:9138-9142.

Southern blot analysis showed three hybridizing EcoRI bands in the humanDNA control that were approximately 12 kb, 11 kb and 5.5 kb in length.Analysis of the somatic cell hybrid DNA indicated that the 12 kb bandwas in two different samples, NA10629, which contained only humanchromosome 6, and NA07299, which contained both human chromosomes 1 andX and, importantly, a portion of chromosome 6 telomeric to p21. The 11kb band was in NA13140, which contains human chromosome 20. The 5.5 kbhybridizing band was found only in sample NA10927A, which containedhuman chromosome 11. PCR/DNA sequencing analysis of these hybrid DNAsamples using primers for cdn-1 or cdn-2, showed cdn-1 sequences inNA10629 (the chromosome 6-containing hybrid DNA) and NA07299 (thechromosome 1, X and 6pter >p21-containing hybrid DNA), indicating thatthe cdn-1 gene resides on chromosome 6, telomeric to p21. cdn-2sequences were found in NA13140, indicating the cdn-2 gene resides onchromosome 20, and cdn-3 sequences were found in NA10927A, indicatingthe cdn-3 gene resides on chromosome 11.

EXAMPLE 8 Modulation of Apoptosis by cdn-1 and cdn-2 in FL5.12 Cells

FL5.12 is an IL-3-dependent lymphoid progenitor cell line (McKearn etal. (1985) Proc. Natl. Acad. Sci USA 82:7414-7418) that has been shownto undergo apoptosis following withdrawal of IL-3 but is protected fromcell death by overexpression of bcl-2. Nunez et al. (1990) J. Immunol.144:3602-3610; and Hockenbery et al. (1990) Nature 348:334-336. Toassess the ability of cdn-1 and cdn-2 to modulate apoptosis, cDNAsencoding cdn-1, cdn-2, two truncated forms of cdn-1 (described below)and bcl-2 were ligated into the mammalian expression vector, pcDNA3(Invitrogen, San Diego, Calif.) and stably introduced into the mouseprogenitor B lymphocyte cell line FL5.12 by electroporation andselection in media containing the antibiotic G418. Assays were thenperformed on bulk transformants as described below.

The effects of the overexpressed genes on FL5.12 cell viability wereexamined at various times following withdrawal of IL-3 and are shown inFIG. 10. Cell viability was assessed by propidium iodide (PI) exclusionon a flow cytometer (Becton Dickinson FACScan). Bcl-2 expressionprotected the cells significantly from cell death while cdn-1 appearedto enhance cell death when compared to the vector control. Cdn-2expression conferred a low level of protection from cell death atearlier times but was insignificant at later time points. Interestingly,cdn-1Δ2 gave a moderate level of protection against cell death.Cdn-1-112, a molecule that contains the N-terminal 112 amino acids ofcdn-1, also appeared to partially protect the FL5.12 cells although atlower levels than Bcl-2.

As shown in Example 7, expression of cdn-1 and cdn-1Δ2 in WIL2 cellsresulted in increased cell survival in response to anti-Fas-mediatedapoptosis and serum withdrawal. Taken together, these data suggest thatthe various cdn molecules are capable of modulating apoptosis in apositive or negative manner, depending on the cell type and apoptoticstimuli. Thus, they are effective in preventing cell death such as inthe post-ischemic reperfusion tissue damage in the heart or in inducingcell death in cells that have escaped apoptotic control, as is the casein various cancers.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1-58. (canceled)
 59. An antisense molecule comprising an oligonucleotideof length sufficient to modulate apoptosis, wherein said antisenseoligonucleotide is complementary to a polynucleotide encoding CDN-1,CDN-2, CDN-3, CDN1Δ1, CDN1Δ2, or CDN1Δ3.
 60. The antisense compound ofclaim 59, which is targeted to a nucleic acid molecule encoding CDN-1and which preferentially inhibits the expression of CDN-1.
 61. Theantisense compound of claim 59, which promotes apoptosis.
 62. Theantisense compound of claim 59, which inhibits apoptosis.
 63. Apharmaceutical composition comprising the antisense molecule of claim 59and a pharmaceutically acceptable carrier or diluent.
 64. A method ofmodulating apoptosis in a cell in a mammal diagnosed as having aproliferative disease, comprising administering to said mammal anantisense oligonucleotide of claim
 59. 65. The method of claim 64,wherein said mammal is a human.
 66. The method of claim 64, wherein saidproliferative disease is cancer.
 67. A method to modulateapoptosis-induced cell death, comprising contacting an endogenouspolynucleotide encoding SEQ ID NO:7 in a cell with an antisensepolynucleotide that inhibits the expression of the polynucleotideencoding SEQ ID NO:7.
 68. The method of claim 67, wherein the antisensepolynucleotide hybridizes under highly stringent conditions to a geneencoding SEQ ID NO:7.
 69. The method of claim 67, wherein the antisensepolynucleotide is fully complementary to a polynucleotide encoding SEQID NO:7.
 70. The method of claim 67, wherein the antisensepolynucleotide is fully complementary to a polynucleotide encoding afragment of SEQ ID NO:7 lacking up to about the first 70 amino acids ofSEQ ID NO:7, wherein the fragment modulates apoptosis in a cell.
 71. Themethod of claim 67, wherein the antisense polynucleotide is fullycomplementary to a polynucleotide encoding a fragment of SEQ ID NO:7truncated after about amino acid position 112, wherein the fragmentmodulates apoptosis in a cell.
 72. The method of claim 67, wherein theantisense polynucleotide is fully complementary to a polynucleotideencoding an amino acid sequence that that is greater than 97% identicalto SEQ ID NO:7, wherein the amino acid sequence modulates apoptosis in acell.
 73. The method of claim 67, wherein apoptosis-induced cell deathis increased in the cell in the presence of the polynucleotide ascompared to in the absence of the polynucleotide.
 74. The method ofclaim 67, wherein the cell displays uncontrolled cell growth.
 75. Themethod of claim 74, wherein the cell is a cancer cell.
 76. The method ofclaim 74, wherein the cell is from a B cell lymphoma.